The demand for increased levels of deoxyribonucleotides during DNA replication requires that ribotide reduction is subject to feedback regulation by the intracellular concentrations of the deoxyribonucleoside triphosphates. Control of ribotide reduction in E. coli is expressed on two levels - through regulation of overall reductase activity and through regulation of which of the four possible ribonucleotide substrates is reduced preferentially. Modulation of the overall activity and specification of the preferred substrate both apparently involve alterations in the degree of association between the proteins constituting the ribotide reductase complex. A quantitative description of these two interrelated, yet distinct, regulatory mechanisms will be studied using sedimentation velocity and equilibrium techniques. This study involves determining the influence of the allosteric effectors and pseudo-substrates on the association state of the ribotide reductase protein B1. The additional presence of the second protein constituting the ribotide reductase system, B2, will be studied as it affects the association state of B1. The existence of direct stable protein-protein interactions the B1 and B2 proteins constituting ribotide reductase and the thioredoxin proteins (consisting of thioredoxin and thioredoxin reductase) also will be studied using a centrifugational approach. This study will attempt to elucidate the means by which electrons are transferred from thioredoxin reductase (linked to NADPH oxidation) to thioredoxin and finally to the ribonucleotide substrate bound to ribotide reductase protein B1. Enzyme for these studies is obtained from an E. coli K strain (W3110) grown under conditions of maximal derepression in growth medium containing limited amounts of thymidine. Subsequent purification of the four proteins involved in ribotide reduction will utilize affinity columns for proteins B1 (dATP-sepharose) and thioredoxin reductase (FAD- sepharose) and antibody columns for protein B2 and thioredoxin.